Staphylococcus aureus mutants resistant to the feed-additive monensin show increased virulence and altered purine metabolism

Abstract

Ionophores are antibacterial compounds that affect bacterial growth by changing intracellular concentrations of the essential cations, sodium and potassium. They are extensively used in animal husbandry to increase productivity and reduce infectious diseases, but our understanding of the potential for and effects of resistance development to ionophores is poorly known. Thus, given their widespread global usage, it is important to determine the potential negative consequences of ionophore use on human and animal health. In this study, we demonstrate that exposure to the ionophore monensin can select for resistant mutants in the human and animal pathogen Staphylococcus aureus, with a majority of the resistant mutants showing increased growth rates in vitro and/or in mice. Whole-genome sequencing and proteomic analysis of the resistant mutants show that the resistance phenotype is associated with de-repression of de novo purine synthesis, which could be achieved through mutations in different transcriptional regulators including mutations in the gene purR, the repressor of the purine de novo synthesis pathway. This study shows that mutants with reduced susceptibility to the ionophore monensin can be readily selected and highlights an unexplored link between ionophore resistance, purine metabolism, and fitness in pathogenic bacteria.IMPORTANCEThis study demonstrates a novel link between ionophore resistance, purine metabolism, and virulence/fitness in the key human and animal pathogen Staphylococcus aureus. The results show that mutants with reduced susceptibility to the commonly used ionophore monensin can be readily selected and that the reduced susceptibility observed is associated with an increased expression of the de novo purine synthesis pathway. This study increases our understanding of the impact of the use of animal feed additives on both human and veterinary medicine.

Keywords: cross-resistance; drug resistance evolution; drug resistance mechanisms; fitness; ionophore; mouse experiment; purine metabolism.

Fig 1

Characterization of monensin-resistant mutants. (A) Whole-genome sequencing analysis was performed for five monensin-resistant mutants. Mutations observed within a single mutant are depicted with the same color. (B) Relative exponential growth rate of the resistant mutants was measured relative to the susceptible S. aureus (growth rate set to 1.0). Four replicates were used in each case, and error bars represent standard deviation. Statistical significance was determined by performing a one-way ANOVA followed by a Tukey HSD test and is denoted as *.

Fig 2

Monensin-induced physiological changes in the susceptible and resistant S. aureus strain. (A) Known effects of monensin on the intracellular ion concentrations are shown for susceptible S. aureus strain, which includes efflux of K⁺ ions and an influx of Na⁺ and H⁺ ions. These changes were experimentally determined for the monensin-resistant and monensin-susceptible S. aureus: (B) changes in intracellular pH, (C) intracellular Na⁺:K⁺ ratio, and (D) intracellular ATP:ADP ratio were determined for the susceptible and four monensin-resistant S. aureus strains. In each case, circles denote measurements in the absence of monensin, and triangles show measurements in the presence of monensin. Two replicates are used in each case, and error bars represent standard deviation.

Fig 3

Whole-cell proteomic analysis of monensin-resistant mutants. (A) A heatmap depicting a subset of proteins with an increased level of protein expression in monensin-resistant mutants, as compared to the susceptible S. aureus. Darker colors represent a higher level of expression. Proteins involved in the de novo purine synthesis pathway are highlighted. Two replicates were used in each case. (B) Expression levels of the different proteins involved in the de novo purine synthesis pathway in the monensin-resistant mutants, relative to the susceptible S. aureus. Expression levels are plotted on a log₂ scale.

Fig 4

Upregulation of de novo purine biosynthesis pathway observed in monensin-resistant mutants. (A) The de novo purine synthesis pathway is depicted with proteins that were upregulated in the monensin-resistant mutants shown in green and those that were downregulated shown in red, both relative to the susceptible S. aureus. (B) The effect of monensin on the susceptible S. aureus is reduced in the presence of xanthine, a metabolite of the purine de novo synthesis pathway. Exponential growth rate and stationary phase densities are plotted for the susceptible S. aureus in the absence of xanthine and monensin, in the presence of xanthine, in the presence of monensin, and in the presence of xanthine and monensin together. Four replicates were used in each case, and error bars represent standard deviation. Statistical significance was determined by performing a two-sided Student’s t-test at P < 0.05 and is denoted as *, while non-significance is shown as ns.

Fig 5

Animal infection experiments to determine the bacterial load of monensin-resistant mutants. (A) Experimental design depicting the infection experiment in mice using monensin-susceptible and monensin-resistant S. aureus. Eight mice were infected by tail injection for each strain. The kidney and spleen from each mouse were then harvested 24 h post-infection, and the bacterial load was determined by plating on TSA plates. (B) Bacterial load in the kidney and spleen are shown for the susceptible (red) and three monensin-resistant (blue) S. aureus strains. A ΔpurR S. aureus mutant (green) was used as a positive control in the experiment. Eight replicates are used in each case, and P-values are calculated for a two-sided Student’s t-test.